Project Summary Cyclic GMP-AMP synthase (cGAS) is a recently discovered enzyme that acts as a foreign DNA sensor to elicit an immune response to pathogens via activation of the STING (stimulator of interferon genes) receptor. Though there are other DNA sensors (e.g., TLR9 ), the cGAS-cGAMP-STING pathway appears to be essential for DNA- mediated immune response irrespective of cell type or DNA sequence. DNA binds to a specific site on cGAS in a non-sequence-dependent manner and activates its catalytic activity, resulting in the production of a unique cyclic nucleotide G(2'-5')pA(3'-5')p (cGAMP) from ATP and GTP precursors. cGAMP binds to the STING receptor with nanomolar affinity and induces expression of type I interferons. Thus, cGAMP plays a fundamental role in human immunity, acting upstream of both T and B cells to trigger an innate immune response. Shortly after its discovery in 2013, aberrant activation of cGAS by self-DNA was shown to contribute to debilitating and sometimes fatal autoimmune diseases, such as systemic lupus erythematosus (SLE), and knocking out cGAS abrogates disease in animal models. The cGAS-STING pathway has also been shown to play a key role in the innate immune response to tumors, and intratumoral injection of cGAMP analogs is an emerging strategy for cancer immunotherapy. Modulating cGAS activity is autoimmunity and cancer. Tumor immunityATP GTP cGAS cGAMP Autoimmune diseases IRF Antimicrobial Immunity 3 STING NF?B Type I IFNs Activation of cGAS by cytoplasmic DNA initiates activation of the innate immune response via induction of Type I IFNs which induce tumor cell specific T cell responses in cancer but induce autoantibodies and cause extensive tissue damage in autoimmune diseases such as SLE. therefore a very compelling therapeutic strategy, both for Though basic research on the cGAS/STING pathway and efforts to target it therapeutically have expanded rapidly, the difficulty in detecting cGAMP has seriously hindered progress on both fronts. It functions at nanomolar concentrations, whereas other nucleotides, including its precursors ATP and GTP, are present at levels as much as 1000-fold higher, making specific detection of cGAMP in cell or tissue extracts extremely challenging. Currently, the only method used is methanol extraction followed by HPLC purification and LC/MS. Similarly, methods for detecting cGAS enzymatic activity involve chromatographic separation of radioactive cGAMP from ATP and GTP. Development of drugs that modulate cGAS will require sensitive, homogenous assay methods for detecting cGAMP with exquisite specificity that can be used for biochemical and cellular HTS assays, and eventually for biomarker assays to support translational research. In Phase I, we produced monoclonal antibodies that recognize cGAMP with more than 1000-fold selectivity vs. other nucleotides and used one of them to develop homogenous detection assays with fluorescent readouts (FP and TR-FRET). We validated the assays as a robust biochemical HTS platform using purified human cGAS; these assays will allow us to begin screening for cGAS modulators under separate grant applications. In Phase II, we will develop more sensitive cGAMP detection reagents and methods that can be used for cellular HTS and biomarker assays. Taking inspiration from recent examples of sandwich-based assays for small molecules, we will use cGAMP antigen design approaches and in vitro evolution to generate a pair of single chain Fv's that bind cGAMP simultaneously with picomolar affinity to allow direct detection of cGAMP in cell and tissue lysates with TR-FRET and/or ELISA assays. The effort will include contributions from two outstanding academic scientists to buttress BellBrook's assay development expertise. Dr. Karl Wittrup, Director of the Koch Institute at MIT and inventor of the yeast display system that we will use for scFv affinity maturation, will act as a consultant for the critical in vitro evolution step. Dr. Keith Elkon, Head of the Division of Rheumatology at University of Washington, one of the world's leading experts on the molecular and genetic basis for autoimmune diseases, who is pioneering efforts to elucidate the role of cGAS/STING in SLE, will collaborate on validation of the biomarker assay with samples from animal models and patients. The development of simple, quantitative cGAMP assays would have very significant scientific and medical impact. The biochemical and cellular cGAMP assays will enable BellBrook and collaborators to pursue HTS- driven efforts to develop first-in-class lead molecules targeting the cGAS/STING pathway for autoimmunity and cancer immunotherapy. More broadly, commercialization of the assays as kits will fill critical gaps in the tools needed for basic cellular and biochemical studies of the cGAS/STING pathway.